Regeneration of biosensors

ABSTRACT

The invention relates to a system for continuous monitoring of analytes in a biological fluid, the system having increased life by virtue of inherent regeneration of sensors employed. An embodiment of the invention includes a biosensor, a sampling device for providing a sample of the biological fluid, device(s) for passing a flow of a background fluid through the flow passage at selectable flow rates, device(s) for injecting the sample into the flow of background fluid, and device(s) for increasing the flow rate of the combined flow. An embodiment of the invention provides device(s) for achieving a washing action at the signal generating portion.

This patent application is a divisional application of U.S. patentapplication Ser. No. 09/462,394 filed on May 3, 2000, now U.S. Pat. No.6,812,031.

The present invention relates in general to the field of biosensors, andin particular to methods and apparatus for regenerating such sensors,thereby increasing the effective life thereof.

In a specific aspect the invention relates to a system for continuousanalysis of analytes in blood or serum comprising means for regenerationof the sensor employed therein.

BACKGROUND OF THE INVENTION

Measurements of analytes in blood is commonly performed by samplingblood from patients and analyzing said samples in a laboratory, oftensituated at a location remote from the ward. E.g. for glucose analysisthere are available special reagent sticks usable for measuring on sitei.e. in the ward. However, the accuracy of such measurements isquestionable, and the error could be 10-20% at best.

Often it is necessary to perform several sequential measurements overperiods of several hours, which is very labor intensive. Furthermore,the risk for errors because of the human intervention is evident, andthe low accuracy is of course also a drawback in this regard.

For the purposes of this application, the term “biosensor” means anydevice having a portion which interacts with biological or biochemicalmaterial, and has the capability to generate a signal indicative of achange in some parameter of said biological or biochemical material as aconsequence of said interaction.

When analytes such as glucose, urea, lactate, ATP, glycerol, creatinineand pyruvate in biological samples, such as blood, plasma or serum areanalyzed using biosensor techniques based on immobilization of enzymes,the sensor surface will be exposed to a certain amount of sample duringa certain time sufficient to achieve an adequate sensor response. It iswell known that the sensor response gradually will degrade because offouling of the surface. This in its turn is a consequence of saidexposure and the interaction between the surface and the substancespresent in the sample that occurs. The chemical and physical compositionof the sample is thereby of importance, the sample i.a. comprising redcells, blood platelets, macromolecules, electrolytes, lipids,red/ox-compounds etc. It is also known that the support material for theenzyme immobilization in biosensors based on enzyme column technology isfouled by the substances present in the sample.

In cases where selective membranes are used for protection of the sensorsurface of biosensors based on enzyme electrode technology, saidmembranes are also fouled by such substances.

This fouling influences the sensor response by substantially reducingthe life and stability of the biosensor.

DESCRIPTION OF RELATED ART

Most known metabolite sensors today are based on the amperometricprinciple, that is measurement of oxygen consumption or hydrogenperoxide production in electrochemical reactions. However, interferencewith reducing/oxidizing substances causes problems like long time drift,need for frequent calibration and short life. Regarding the samplingprocedure there exist devices which, before the actual measurement,condition the blood before it enters the actual sensor by e.g.introducing a special step, such as dialysis. This is both a morecomplicated solution and also more expensive, since the dialysiscassette has to be replaced before a new measurement can be made.

Another known sensor principle is by utilizing the heat production whenthe analytes are decomposed by the appropriate enzyme for the analyte inquestion. This so called enzyme calorimeter principle is known from U.S.Pat. No. 4,021,307. The enzyme calorimeter disclosed therein is howevernot suited for direct measurement on whole blood, since the blood cellsquickly will clog the column containing the immobilized enzyme, due toadsorption of blood constituents such as various cells, trombocytes,proteins etc. This effect could be circumvented to a certain extent bydiluting the blood at least ten times, which will reduce the sensitivityof the measurement considerably. However such a measure would require anextra supply of a diluting solution. Another way of reducing theclogging of the column is to use a special super porous support materialwith a pore size larger than 10 μm. This support made from agarose, ishowever softer than the conventional support materials used in thisfield, preferably glass, and therefore are at a certain risk of(occasionally) being compressed by the blood sample, which in turnquickly will clog the column.

Thus, at present there is no reliable method and system available forthe direct and continuous analysis of whole blood drawn from patients.

SUMMARY OF THE INVENTION

The present invention therefore seeks to provide an improved method ofanalyzing whole blood in respect of analytes such as glucose, lactate,urea, ATP, glycerol, creatinine and pyruvate wherein the drawbacks ofthe prior art methods are alleviated.

In particular the active life of a biosensor that is used for suchanalysis is prolonged by providing for reduced fouling of the sensor byregenerating the sensor in accordance with the invention.

The method according to the invention is defined in claim 1.

In a second aspect of the invention there is also provided a system forlong time measurements of whole blood directly and continuously sampledfrom a patient, wherein the flow of the sampled blood is held at a verylow rate.

The system according to the invention is defined in claim 12.

Further scope of applicability of the present invention will becomeapparent from the detailed description given hereinafter.

However, it should be understood that the detailed description andspecific examples, while indicating preferred embodiments of theinvention, are given by way of illustration only, since various changesand modifications within the spirit and scope of the invention willbecome apparent to those skilled in the art from this detaileddescription.

The present invention will become more fully understood from thedetailed description given hereinbelow and the accompanying drawingswhich are given by way of illustration only, and thus not limitative ofthe present invention, and wherein

FIG. 1 is an overview of a system according to the invention;

FIG. 2 is a cross sectional view through a connector according to theinvention;

FIG. 3 is a view similar to FIG. 2, but showing a conventional connectorwithout the inventive sealing feature; and

FIG. 4 is a flow chart illustrating the sequence of steps in the methodof the invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

In FIG. 1 there is disclosed a system for performing a continuousmonitoring of the concentration of analytes in blood.

It comprises a blood sampling device 2, such as a cannula 4 inserted ina vein of a patient. The cannula 4 is connected to the other systemcomponents via a tubing 8 and a suitable connector 6 (to be described)connecting to a pump 10, which is provided for drawing the variousfluids through the system at controlled flow rates. The pump is amultichannel pump, and has a first input 12 for the blood from thesampling device 2, a second input 14 for buffer solution from a bufferstorage 15, and a third input 16 for anticoagulant from an anticoagulantreservoir 17. Anticoagulant is fed through a line 9 into the sample flowin line 8 at a point near the tip of the catheter 4.

Alternatively there may be provided separate pumps for the variouscomponents.

The valve 20 is important for the operation in accordance with theinvention, and has two inputs, one input 21 for sample blood, fed frompump 10 through line 22, and one input 23 for buffer, fed through line24. There are also two outputs, a first connecting to line 25 a feedingthe fluid to the analysis portion, and a second connecting to line 25 bfor discharging the fluid as waste. The valve 20 is designed such as topermit fluid (i.e. blood in this embodiment) from line 22 to be injectedinto the buffer flow from line 24.

All surfaces in the system exposed to sample are coated with heparin inorder to make the system blood compatible.

The actual sample analysis may be carried out in a so called enzymereactor (ER) 26, although it is contemplated that any biosensor type maybe used, provided it has a sensitive portion arranged in some kind offlow passage where flow past the sensitive portion of the sensor can becontrolled. Thus, a sensor of a type that is merely immersed in a liquidwould not be suitable for use with this invention. An ER is used in apreferred embodiment and will be described in more detail below.

The system also comprises a control unit 50, which may be a microprocessor or a PC. An interface 55 is connected between the control unit50 and the components in the system, such that control signals to thepump 10 and the valve 20 are fed via lines 52 and 54 respectively. Thus,the pumping rate in the various independently operated flow passages maybe increased or decreased, and the valve 20 may be switched between itsvarious positions by commands issued by the control unit in response tosignals from the biosensor. An amplifier 56 is provided for amplifyingthe signals from the biosensor 26, and feeding said signals to theinterface on line 58, for further transmission to the control unit whichuses the information thus obtained to issue the appropriate controlcommands to the pump and valve.

The Enzyme Reactor

An enzyme reactor (ER) 26 comprises a sensor column. The column containssupport material such as beads of glass or hard polymer resin, on whichenzyme has been immobilized. Immobilization of enzyme is standardprocedure and does not form part of this invention, and will hence notbe described in detail herein.

The operation and function of the ER 26 is as follows.

The sensor column has two thermistors 30, 32, one 30 arranged at thecolumn inlet and the other 32 at the column outlet. Fluid entering thecolumn will begin reacting with the enzyme that is immobilized on thebeads in the column, and will thereby generate heat, causing thetemperature of the fluid to increase. By monitoring the temperatures atthe inlet and outlet respectively, and integrating the temperature overtime, the integral obtained will correspond to the heat of reaction,which then may be related to the concentration of e.g. glucose in thefluid.

Because of non-specific reactions that may be exothermic or endothermicand which occur in the column, temperature fluctuation must be accountedfor. Thermostating the reactor is one way of doing this, but it can beachieved also in other ways and by other means, and is not crucial tothe invention.

Operation

Returning now to FIG. 1, there is illustrated an embodiment of thesystem which comprises a biosensor 26 arranged in a thermostatedenvironment. The system is operated as follows:

The catheter 4 is inserted in a blood vessel of a patient, and connectedto the tubing of the system by means of a connector 6 (to be described).Initially the pump will draw blood through line 8 via line 22, throughthe valve 20 and to the waste line 25 b for disposal, and buffer frombuffer storage 15 is drawn via line 18 through the valve 20 and via line25 a into the enzyme reactor 26. The continuously measured signalsobtained from the sensor when the buffer passes through it will form abackground or zero level, and the flow of buffer will be referred to asa “background flow” in this application. The rate of background flow mayvary in the range 0.1-10 ml/min., and preferably is 1 ml/min.

If desired, and indeed it is mostly required, anticoagulant is mixedwith the blood. Normally a ratio between sample and anticoagulant of 1:1will be used, although other ratios are conceivable for specificconditions. The anticoagulant is pumped in line 9 and injected in thesample flow line 8 near the catheter 4 tip.

At a time when it is desired to make a measurement the valve is given a“SWITCH TO INJECTION MODE” command to the effect that the blood isredirected into the buffer stream, for a period of time of a durationsufficient for an aliquot of 10 μl to be entered as a liquid plug in thebuffer stream (other sample volumes may of course be employed, but atthe present time 10 μl has proven suitable in most cases). This “bloodplug” is passed in line 25 a, which runs in the thermostated medium,where the sample obtains a controlled temperature, and then it entersthe ER 26.

As soon as the blood, containing e.g. glucose, reaches the ER 26, theglucose will start reacting with the enzyme, thereby evolving heat ofreaction. The enzyme reaction is a very rapid process. Other componentsin the blood, such as various cells, trombocytes and proteins having atendency to adsorb to the material inside the reactor, will begin toadsorb. The latter process is however a slow process compared to thediffusion controlled enzyme reaction. The small glucose moleculesdiffuse very much faster than the macromolecules and other macrocomponents in the blood.

The thermistor 32 at the output end of the ER 26 will experience a risein temperature caused by the enzyme reaction occurring in the reactor(at this time the entire sample preferably should have entered thereactor, although this is not absolutely necessary, as will be discussedbelow).

In the present embodiment the temperature increase sensed by thermistor32 is transmitted to the control unit which is programmed to respond toan increase in the temperature signal to issue a INCREASE BUFFER FLOWRATE command to the pump 10 to increase the rate of flow of the bufferby 5-100%, preferably by 10-50%, most preferably by 15-30%.

By balancing the flow rates, i.e. defining a suitable ratio betweenbackground flow rate and increased flow rate, it is possible to create asituation where larger components, such as cells, proteins etc, arewashed away before they have had an opportunity to adsorb on the activesurfaces inside the ER 26, and at the same time allow the smallermolecules of interest sufficient time to react with the enzyme to suchan extent that it is possible to detect the reaction.

This balancing of flow ratios within the given limits is made bystraight forward routine experimentation for a given system, and iseasily done by the skilled man.

In an alternative embodiment it may be sufficient if only a fraction ofthe sample has entered the reactor. In this case the detection of signalonset is not used for triggering. Instead a certain time is determinedempirically, namely the time it takes for the sample to just about reachthe reactor after injection into the background flow. This time is thenprogrammed into the control unit and used as a starting point forincreaseded flow. This time can of course be selected such thatdifferent fractions of sample enter the reactor. It should be noted thatif only a very minor fraction has entered when increaseded flow isinitiated, the signal will be low; however, in most cases the entiresample will have reached the reactor by virtue of the void volume of thereactor being substantially larger than the sample volume.

It could also be possible to wait a short time, such as up to 5 seconds,after the sample completely has entered the reactor, i.e. after thedetection by thermistor 32, before increasing the flow. Thus, in factthere is a time interval during which increased flow can be performed.The actual set of parameters has to be found empirically for eachindividual system, and the skilled man will be able to find theseparameters without inventive work.

The flow pulse at the higher flow rate is maintained until a preselectedsignal value from the reaction response has been recorded, e.g. a peakmaximum, and at this point the flow will be decreased by a RETURN TONORMAL FLOW command to the pump 10, thereby stopping the additional flowof buffer solution. The duration of the pulse of increased flow rate maybe 10-60 s, preferably 20-40 s.

Temperature fluctuations may be eliminated by thermostating the system,e.g. having the ER 26 immersed in a controlled temperature bath.

FIG. 4 illustrates the control algorithm in a simplified flow chartform.

When it is desired to make a measurement, the operator may select aINJECT command from a menu, or the computer may be programmed to issuethe command at a preselected point in time. This command will set thevalve such that the flow of sample (blood) is diverted into the bufferflow, for a time sufficient to inject the desired sample volume, i.e. 10μl. Then, the valve is reset to normal mode, i.e. the blood isdischarged as waste.

If the system parameters, such as configurations, flows etc. are welldefined, then it is possible to preset the time T when increased flow isto be initiated. Thus, when the elapsed time t after injection of sampleequals T, increasing is initiated by the computer.

Alternatively, the computer continuously registers the signal from thethermistor, and when a signal gradient, i.e. a temperature rise, ofsufficient magnitude occurs, increasing the flow is initiated.

The increase in flow is performed by increasing the pump speed, by thecomputer issuing a INCREASE PUMP SPEED command to the interface, suchthe initial pump rate P₀ is increased by a factor corresponding to anincrease of 5-100%, preferably by 10-50%, most preferably by 15-30%.Thus, the pump speed during increased flow (or pulse) mode isP=P ₀ +XP ₀.

After a time Δt when the reaction in the reactor is complete, the pumpspeed is reverted back to the initial value P₀.

Then, control reverts to the computer for either a programmed newmeasurement at a preselected point in time, or an operator initiatedmeasurement.

The Connector

In FIG. 2 a connector device for connecting to a patient, suitable foruse in connection with the system according to the invention isillustrated.

The connector device, generally designated 100, comprises a male part,generally designated 102, and a female part, generally designated 104.

The male part 102 is provided on the distal end of a catheter 106 thathas been inserted in e.g. a blood vessel of a patient.

The female part 104 comprises a narrow tube 108 of e.g. steel, the innerdiameter of which is larger than the inner diameter of the lumen 110 ofthe catheter 106. The ratio between diameters is preferably 2-3:1.Furthermore, the steel tube 108 is milled or ground on its outerproximal end such that a sharp cutting edge 112 is formed, i.e. theouter surface is made slightly conical at the proximal end.

The tube 108 is inserted in and fixed centrally of a concentric socketstructure 114, forming said female part 104. The socket 114 thuscomprises a cylinder like element having a circular/cylindrical openingor bore 116, the inner surface of which is slightly tapered, and in thecenter of which the tube 108 protrudes a fractional distance of thedepth of said opening. Thus the cutting edge 112 of the tube 108 islocated somewhere in the region between the bottom 118 of said opening116 and its peripheral edge 120. The tapering is such that the diameterat the bottom 118 is slightly smaller than the diameter at theperipheral edge 120.

Similarly, the catheter is located centrally of a cylindrical member 122forming the male part 102, the outer diameter of which snugly fitsinside the opening of the female part 104. The end surface 124 of thecatheter 106 is flush with the end surface 126 of the cylindrical member122. The tube 108 extends so much away from the bottom 118 of the femalepart that when the male and female parts are connected, the sharp edge112 will penetrate the end surface of the catheter 106.

The catheter 106 is made of a material that is enough resilient or soft,that when the male and female parts are connected, the cutting edge 112sinks into the end surface 124 of the catheter 106. Thereby a reliableand safe connection is provided in the transport of blood from thepatient to the measuring system. Suitable materials for the catheter aree.g. soft PVC/silicone.

An example of a suitable locking device usable with the connector andhaving a male/female structure as outlined above is a Luer®-type lock.

As can be seen in the figure there is an abrupt change in the flowcross-section at the connection between catheter 106 and tube 108. Thisis essential in the sense that it will prevent or alleviate clogging ofthe flow path. This principle is known.

By providing the direct contact connection between tube 108 and catheter106, as disclosed above, the large volume 116 is eliminated from theflow path. This is important in the sense that if the blood would haveto pass such a large volume before entering the tube 108, there would beenough time for the constituents of the blood to adhere to the innersurfaces of said volume 116. In FIG. 3 a connector comprising anordinary Luer-lock type coupling is shown. The male part 102 and thefemale part 104 are connected such as to form a dead space 119. It isself evident that the flow rate will decrease drastically when the bloodenters the large dead space 119 inside the coupling, thereby giving theblood constituents time to adhere to the inner surface of the connectorand eventually clog the connector.

Of course it is equally conceivable to provide the catheter in thefemale part and the tube in the male part. However, the first embodimentis preferred since the sharp edge of the tube will be protected ifarranged “inside” the female part, as shown.

Other types of couplings are of course conceivable, the importantfeature is the provision of a sharp edge on the tube, and a catheterhaving the necessary softness or resiliency that the edge will actuallysink into the material when the parts are connected.

For example one could envisage some type of screw and nut connector, ora bayonet type coupling.

The invention will now be further illustrated by way of the followingnon-limiting Examples.

EXAMPLES

The following Examples were performed with the setup shown in FIG. 1.The sensor was an enzyme reactor having dimensions 20 mm length×4 mmdiameter.

The skilled man will easily be able to select suitable thermistorshaving the appropriate properties. One example of thermistor is obtainedfrom Victory Eng. Inc.

The sample volume was 5 or 10 μl, and the background flow was 1 ml/min.

Signal values are given in Volts.

Example I (comparative)

In this example the flow was kept constant, and thus no increased flowwas applied. The sample (blood) volume was 5 μl, and the base linesignal was recorded before and after detection was made. Threeconsecutive runs were performed.

Signal/V

Sample No. Before det. After det. 1 0.10 0.15 2 0.15 0.23 3 0.22 0.34

As is clearly demonstrated the baseline signal before detectionincreases from 0.10 to 0.22 V, and also the baseline signal afterdetection increases from 0.15 to 0.34 V.

Example II (comparative)

The experiment of Example I was repeated with a fresh sensor and newsamples.

Sample No. Before det. After det. 4 0.14 0.38 5 0.35 0.63

Again the base line signals clearly are not reproducible between runs.

Example III (comparative)

In this example the flow was also kept constant but a sample preparedfrom a standard solution and glucose was introduced, and passed throughthe reactor.

Sample No. Before det. After det. 6 0.30 0.31 7 0.29 0.31

As can be seen, the base line signal is not affected.

Example IV (comparative)

The same conditions as in Example I, but the sample volume is increasedto 10 μl.

Sample No. Before det. After det.  8 0.55 0.64  9 0.59 0.67 10 0.62 0.70

Again, the base line is not reproducible between runs.

Example V (According to the Invention)

In this example the sample volume was 10 μl. When the onset of sensorresponse was detected, the buffer flow was increased by 15% andmaintained at that level for 20 seconds, when the response signal beganto decrease again.

Sample No. Before det. After det. 11 0.23 0.22 12 0.23 0.22 13 0.23 0.2214 0.22 0.22 15 0.21 0.22 16 0.20 0.22 17 0.21 0.22 18 0.21 0.21 19 0.230.21

As can be seen from the table, 9 consecutive runs were made and the baseline returned reproducibly to the same level within the accuracy thatmeasurements allow.

The system described herein is preferably designed as a “bed-sidemonitor”, i.e. a portable system for making around-the-clocksurveillance of e.g. intensive care patients, or patients undergoingdialysis.

The control unit and other hardware components are thereby integrated inone single piece of equipment that is easily moved from one location toanother.

Although the description has been made with reference to a system andmethod for analyzing analytes in blood, it is equally possible to usethe inventive ideas for other types of complex biological/biochemicalmedia, such as fermentation media, animal cell culture media.

For example it is suitable for analyzing ethanol or residual sugar inmash in brewing processes. It could also be used for analyzing varioussubstances, e.g. insulin, amino acids or growth hormone in cell culturemedia.

It could also be used to analyze various components in milk or similarfoodstuffs.

The skilled man could envisage numerous other applications of the basicprinciple of the invention, and implement them without inventive work.

Such variations are not to be regarded as a departure from the spiritand scope of the invention, and all such modifications as would beobvious to one skilled in the art are intended to be included within thescope of the following claims.

1. A system for continuous monitoring of analytes in a biological fluid,the system having increased life by virtue of inherent regeneration ofsensors employed, the system comprising: a) a biosensor of the typehaving a flow passage through which fluid is being passed at selectableflow rates, and a sensitive element located in said flow passage whichis responsive to some component or property of a biological fluid; b)means for providing a signal from said sensitive element; c) a samplingdevice for providing a sample of said biological fluid; d) means forpassing a background flow through said flow passage at controlled flowrates; e) means for injecting said sample into said background flow atselectable points in time; f) means for increasing the flow rate of saidbackground flow during passage of the sample through said flow passagein order to achieve a washing action on the signal generating portion,wherein the means comprises a control unit programmed to respond tosignals from said biosensor.
 2. The system of claim 1, wherein saidsampling device comprises a catheter insertable in a blood vessel of ahuman or an animal, and tubing connecting the catheter to the system. 3.The system of claim 1, wherein said means for passing a background flowthrough said flow passage at controlled flow rates comprises a pump andappropriate tubing.
 4. The system of claim 1, wherein said means forinjecting said sample into said background flow comprises a valveswitchable between injection and waste disposal modes.
 5. The system ofclaim 1, wherein said means for providing a signal from said signalgenerating portion comprises at least one thermistor.
 6. The system ofclaim 1, further comprising a connector, for connecting said samplingdevice to said means for passing a background flow through a controlpassage at controlled flow rates, the connector comprising: a male and afemale part; a tube of a hard material such as steel having an innerdiameter, and being inserted in the center of one of said male andfemale parts and protruding from an end surface of said part; a catheterof a soft material inserted in the center of the other of said male andfemale parts and having an inner diameter substantially smaller than theinner diameter of said tube, and having an essentially flat end surface,wherein the protruding end of said tube is ground such as to form asharp circumferential edge, and wherein the positions of said tube andsaid catheter in their respective male or female part are such that whensaid male and female parts are connected, said sharp edge penetratesinto said catheter, thereby forming a fluid tight connection.